The goal of this project is to develop suppressor mutations in mammalian cells. Our approach is to obtain suppressible mutants in the thymidine kinase (TK) gene of herpes simplex virus (HSV) to use as a screening device for such host cells. During the current year we have developed two in vitro protein synthesis systems which make HSV proteins. With our previously isolated TK mutants we have tested this system and have shown that the TK protein is made in vitro and that we get synthesis of mutant TK (shortened polypeptides) when the system is programmed with mutant mRNA. Once we know which mutants are suppressible in vitro, we will use these to screen clones of cells grown from mutagenized stocks. By growing relatively few clones per dish, and then infecting with the proper amount of TK minus virus in the presence of BudR, the infected clones which are su minus will not suppress the TK minus phenotype, i.e., BudR resistance. However, the rare su plus clone when infected with TK minus virus will suppress the TK mutant and the infection will be blocked by the sensitivity to BudR, which results from the suppressed TK phenotype. It is relatively simple, then, to screen many clones for the absence of viral infection (plaques). Once such clones are found, the in vitro protein synthesis system will again be used in studying the mechanism involved in the suppression.